The mechanisms by which active neurons, via astrocytes, rapidly signal intracerebral arterioles to dilate remain obscure. Here we show that modest elevation of extracellular potassium (K+) activated inward rectifier K+ (Kir) channels and caused membrane potential hyperpolarization in smooth muscle cells (SMCs) of intracerebral arterioles and, in cortical brain slices, induced Kir-dependent vasodilation and suppression of SMC intracellular calcium (Ca2+) oscillations. Neuronal activation induced a rapid (<2 s latency) vasodilation that was greatly reduced by Kir channel blockade and completely abrogated by concurrent cyclooxygenase inhibition. Astrocytic endfeet exhibited large-conductance, Ca2+-sensitive K+ (BK) channel currents that could be activated by neuronal stimulation. Blocking BK channels or ablating the gene encoding these channels prevented neuronally induced vasodilation and suppression of arteriolar SMC Ca2+, without affecting the astrocytic Ca2+ elevation. These results support the concept of intercellular K+ channel-to-K+ channel signaling, through which neuronal activity in the form of an astrocytic Ca2+ signal is decoded by astrocytic BK channels, which locally release K+ into the perivascular space to activate SMC Kir channels and cause vasodilation.

BackgroundMicroglia cells continuously survey the healthy brain in a ramified morphology and, in response to injury, undergo progressive morphological and functional changes that encompass microglia activation. Although ideally positioned for immediate response to ischemic stroke (IS) and reperfusion, their progressive morphological transformation into activated cells has not been quantified. In addition, it is not well understood if diverse microglia morphologies correlate to diverse microglia functions. As such, the dichotomous nature of these cells continues to confound our understanding of microglia-mediated injury after IS and reperfusion. The purpose of this study was to quantitatively characterize the spatiotemporal pattern of microglia morphology during the evolution of cerebral injury after IS and reperfusion.MethodsMale C57Bl/6 mice were subjected to focal cerebral ischemia and periods of reperfusion (0, 8 and 24 h). The microglia process length/cell and number of endpoints/cell was quantified from immunofluorescent confocal images of brain regions using a skeleton analysis method developed for this study. Live cell morphology and process activity were measured from movies acquired in acute brain slices from GFP-CX3CR1 transgenic mice after IS and 24-h reperfusion. Regional CD11b and iNOS expressions were measured from confocal images and Western blot, respectively, to assess microglia proinflammatory function.ResultsQuantitative analysis reveals a significant spatiotemporal relationship between microglia morphology and evolving cerebral injury in the ipsilateral hemisphere after IS and reperfusion. Microglia were both hyper- and de-ramified in striatal and cortical brain regions (respectively) after 60 min of focal cerebral ischemia. However, a de-ramified morphology was prominent when ischemia was coupled to reperfusion. Live microglia were de-ramified, and, in addition, process activity was severely blunted proximal to the necrotic core after IS and 24 h of reperfusion. CD11b expression, but not iNOS expression, was increased in regions of hyper- and de-ramified microglia during the course of ischemic stroke and 24 h of reperfusion.ConclusionsOur findings illustrate that microglia activation after stroke includes both increased and decreased cell ramification. Importantly, quantitative analyses of microglial morphology and activity are feasible and, in future studies, would assist in the comprehensive identification and stratification of their dichotomous contribution toward cerebral injury and recovery during IS and reperfusion.

An increase in CO(2)/H(+) is a major stimulus for increased ventilation and is sensed by specialized brain stem neurons called central chemosensitive neurons. These neurons appear to be spread among numerous brain stem regions, and neurons from different regions have different levels of chemosensitivity. Early studies implicated changes of pH as playing a role in chemosensitive signaling, most likely by inhibiting a K(+) channel, depolarizing chemosensitive neurons, and thereby increasing their firing rate. Considerable progress has been made over the past decade in understanding the cellular mechanisms of chemosensitive signaling using reduced preparations. Recent evidence has pointed to an important role of changes of intracellular pH in the response of central chemosensitive neurons to increased CO(2)/H(+) levels. The signaling mechanisms for chemosensitivity may also involve changes of extracellular pH, intracellular Ca(2+), gap junctions, oxidative stress, glial cells, bicarbonate, CO(2), and neurotransmitters. The normal target for these signals is generally believed to be a K(+) channel, although it is likely that many K(+) channels as well as Ca(2+) channels are involved as targets of chemosensitive signals. The results of studies of cellular signaling in central chemosensitive neurons are compared with results in other CO(2)- and/or H(+)-sensitive cells, including peripheral chemoreceptors (carotid body glomus cells), invertebrate central chemoreceptors, avian intrapulmonary chemoreceptors, acid-sensitive taste receptor cells on the tongue, and pain-sensitive nociceptors. A multiple factors model is proposed for central chemosensitive neurons in which multiple signals that affect multiple ion channel targets result in the final neuronal response to changes in CO(2)/H(+).

Abstract-Neuronal activity in the brain is thought to be coupled to cerebral arterioles (

The arcuate nucleus (ARC) of the hypothalamus plays a key role in sensing metabolic feedback and regulating energy homeostasis. Recent studies revealed activation of microglia in mice with high-fat diet (HFD)-induced obesity (DIO), suggesting a potential pathophysiological role for inflammatory processes within the hypothalamus. To further investigate the metabolic causes and molecular underpinnings of such glial activation, we analyzed the microglial activity in wild-type (WT), monogenic obese ob/ob (leptin deficient), db/db (leptin-receptor mutation), and Type-4 melanocortin receptor knockout (MC4R KO) mice on either a HFD or on standardized chow (SC) diet. Following HFD exposure, we observed a significant increase in the total number of ARC microglia, immunoreactivity of ionized calcium binding adaptor molecule 1 (iba1-ir), cluster of differentiation 68 (CD68-ir), and ramification of microglial processes. The ob/ob mice had significantly less iba1-ir and ramifications. Leptin replacement rescued these phenomena. The db/db mice had similar iba1-ir comparable with WT mice but had significantly lower CD68-ir and more ramifications than WT mice. After 2 weeks of HFD, ob/ob mice showed an increase of iba1-ir, and db/db mice showed increase of CD68-ir. Obese MC4R KO mice fed a SC diet had comparable iba1-ir and CD68-ir with WT mice but had significantly more ramifications than WT mice. Intriguingly, treatment of DIO mice with glucagon-like peptide-1 receptor agonists reduced microglial activation independent of body weight. Our results show that diet type, adipokines, and gut signals, but not body weight, affect the presence and activity levels of hypothalamic microglia in obesity.

Angiotensin II-mediated vascular brain inflammation emerged as a novel pathophysiological mechanism in neurogenic hypertension. However, the precise underlying mechanisms and functional consequences in relation to blood brain barrier integrity and central angiotensin II actions mediating neurohumoral activation in hypertension are poorly understood. Here, we aimed to determine whether blood brain barrier permeability within critical hypothalamic and brainstem regions involved in neurohumoral regulation was altered during hypertension. Using digital imaging quantification following intravascularly injected fluorescent dyes and immunohistochemistry, we found increased blood brain barrier permeability, along with altered key blood brain barrier protein constituents, in spontaneously hypertensive rats within the hypothalamic paraventricular nucleus, the nucleus of the solitary tract, and the rostral ventrolateral medulla, all critical brain regions known to contribute to neurohumoral activation during hypertension. Blood brain barrier disruption, including increased permeability and down-regulation of constituent proteins, was prevented in spontaneously hypertensive rats treated with the AT1 receptor antagonist Losartan, but not with hydralazine, a direct vasodilator. Importantly, we found circulating angiotensin II to extravasate into these brain regions, co-localizing with neurons and microglial cells. Taken together, our studies reveal a novel angiotensin II-mediated feed-forward mechanism during hypertension, by which circulating angiotensin II evokes increased blood brain barrier permeability, facilitating in turn its access to critical brain regions known to participate in blood pressure regulation.

Background-In obesity, the excessive synthesis of aldosterone contributes to the development and progression of metabolic and cardiovascular dysfunctions. Obesity-induced hyperaldosteronism is independent of the known regulators of aldosterone secretion, but reliant on unidentified adipocyte-derived factors. We hypothesized that the adipokine leptin is a direct regulator of aldosterone synthase (CYP11B2) expression and aldosterone release and promotes cardiovascular dysfunction via aldosterone-dependent mechanisms. Methods and Results-Immunostaining of human adrenal cross-sections and adrenocortical cells revealed that adrenocortical cells coexpress CYP11B2 and leptin receptors. Measurements of adrenal CYP11B2 expression and plasma aldosterone levels showed that increases in endogenous (obesity) or exogenous (infusion) leptin dose-dependently raised CYP11B2 expression and aldosterone without elevating plasma angiotensin II, potassium or corticosterone. Neither angiotensin II receptors blockade nor α and β adrenergic receptors inhibition blunted leptin-induced aldosterone secretion. Identical results were obtained in cultured adrenocortical cells. Enhanced leptin signaling elevated CYP11B2 expression and plasma aldosterone, whereas deficiency in leptin or leptin receptors blunted obesity-induced increases in CYP11B2 and aldosterone, ruling out a role for obesity per se. Leptin increased intracellular calcium, elevated calmodulin and calmodulinkinase II expression, whereas calcium chelation blunted leptin-mediated increases in CYP11B2, in adrenocortical cells. Mineralocorticoid receptor blockade blunted leptin-induced endothelial dysfunction and increases in cardiac fibrotic markers. Conclusions-Leptin is a newly described regulator of aldosterone synthesis that acts directly on adrenal glomerulosa cells to increase CYP11B2 expression and enhance aldosterone production via calcium-dependent mechanisms. Furthermore, leptin-mediated aldosterone secretion contributes to cardiovascular disease by promoting endothelial dysfunction and the expression of profibrotic markers in the heart.

Alterations in the partial pressure of carbon dioxide (P CO 2 ) and pH are known to change the activity of specialized central neurones referred to as chemosensitive neurones. We define a chemosensitive neurone as one whose firing rate is increased by an increase in CO 2 /H + . Such neurones have been localized to a number of brainstem centres involved in a variety of physiological functions including the regulation of cardiovascular and respiratory systems. These centres include the ventral medullary surface, the ventrolateral medulla (VLM), the solitary tract (NTS), the medullary raphe and the locus coeruleus (LC) (Nattie, 1995(Nattie, , 1998a(Nattie, ,b, 1999Richerson, 1995;Ritucci et al. 1998). The last of these regions, the LC, is located in the dorsal pons of the brainstem. It is the nucleus in the central nervous system with the highest number of noradrenergic neurones and has been associated with a number of physiological and behavioural functions including the sleep-wake cycle, feeding, cardiovascular and respiratory control, nociception, attention and learning (Hobson et al. 1975;Aston-Jones, 1985;Oyamada et al. 1998 Oyamada et al. 1998). The high degree of chemosensitivity makes this nucleus an ideal one for the study of the cellular pathways by which an increase in CO 2 /H + leads to an increase in neuronal discharge.Increased neuronal firing rate in response to high CO 2 /H + could be brought about by changes in three possible signals: extracellular pH (pH o ), intracellular pH (pH i ) or molecular CO 2 . There is evidence indicating that a change in pH i (intracellular acidification), rather than in pH o or molecular CO 2 , is the major signal responsible for the increase in firing rate seen in chemosensitive neurones (Lassen, 1990;Putnam, 2001;. Most cells respond to intracellular acidification with pH i recovery due to activation of pH-regulating membrane transporters. However, several studies have shown that neurones from various chemosensitive regions do not exhibit pH i recovery from intracellular acidification when pH o is also reduced (Ritucci et al. 1997;Putnam, 2001;), e.g. in response to hypercapnic acidosis (HA). For instance, neurones from the NTS and the VLM show sustained intracellular acidification without pH i recovery in response to hypercapnic acidosis _ , pH o 6.8) resulted in a slow intracellular acidification to a maximum fall of about 0.26 pH units and a 72 % increase in firing rate. For all treatments, the changes in pH i preceded or occurred simultaneously with the changes in firing rate and were considerably slower than the changes in pH o . In conclusion, an increased firing rate of LC neurones in response to acid challenges was best correlated with the magnitude and the rate of fall in pH i , indicating that a decrease in pH i is a major part of the intracellular signalling pathway that transduces an acid challenge into an increased firing rate in LC neurones.